Cerebral blood vessels from several species are innervated by vasodilator nerves. Acetylcholine (ACh) released from parasympathetic cholinergic nerves was first suggested to be the transmitter for vasodilation. However, direct effect of ACh on cerebral vascular smooth muscle is constriction, not dilation. The exact transmitter role of ACh in regulating cerebral vascular tone therefore remains unclear. Convincing evidence has been presented to indicate that nitric oxide (NO) plays a predominant role in cerebral neurogenic vasodilation. Nitrergic (NOergic) nerves have been shown to originate in the sphenopalatine ganglion (SPG). Choline acetyltransferase (ChAT) and NO synthase (NOS) are colocalized in the same neurons in the SPG and in cerebral perivascular nerves. It has been suggested that ACh acts presynaptically to inhibit release of co-transmitter NO. Indirect evidence further suggested that presynaptic muscarinic M2-receptors on cerebral perivascular NOergic nerves mediated inhibition of NO release from these nerves. The inhibition was thought due primarily to a decreased Ca2+ influx through N-type Ca2+ channels on NOergic nerves. Direct evidence supporting this concept has not been presented. Our hypothesis is that ACh acts like a presynaptic transmitter in negative modulation of release of the primary transmitter NO, resulting in decreased neurogenic vasodilation. The proposed study is designed to investigate the role of presynaptic muscarinic M2-receptors in modulating voltage-dependent N-type Ca2+ channels in cerebral neurogenic vasodilation. In vitro tissue bath techniques, techniques of electrophysiological and chemical analysis, and light and ultrastructural immunocytochemistry will be utilized to provide a comprehensive and multifaceted approach to the problem. We plan to use isolated cerebral arteries and cultured SPG of the pig and rat to examine: a) a presynaptic muscarinic M2 receptor subtype- mediated inhibition of cerebral NOergic vasodilation a general phenomenon among major cerebral arteries? b) do presynaptic muscarinic M2 receptors mediate inhibition of endogenous NO release in cerebral perivascular nerves? c) do presynaptic muscarinic M2 receptors mediate inhibition of N-type Ca2+ channels in cultured SPG? d) possible mechanisms involved in muscarinic M2 receptor-mediated inhibition of N-type Ca2+ channels in cultured SPG; e) immunohistochemical characterization of transmitter substances in cultured SPG; and f) colocalization of muscarinic M2 receptors and N-type Ca2+ channels, and other transmitter substances in cultured SPG neurons. Results of these studies will provide fundamental information and new insight for establishing a potential functional role of parasympathetic innervation and presynaptic regulation of NO release by classical "transmitters" in regulating cerebral vascular tone. This research is a step toward our long-term goal to define the transmitter mechanisms in cerebral vasodilation and constriction, and their alterations in cerebral vascular diseases.